本文選題：海馬神經(jīng)元　切入點(diǎn)：原子力顯微技術(shù)(AFM)　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的：利用原子力顯微鏡技術(shù)(AFM)和轉(zhuǎn)基因技術(shù)研究原代培養(yǎng)的海馬神經(jīng)元表面超微結(jié)構(gòu)、相互間的連接結(jié)構(gòu)及其相關(guān)功能。材料和方法：取新生12小時(shí)以內(nèi)的C57小乳鼠,在無菌條件下原代培養(yǎng)海馬神經(jīng)元細(xì)胞。選擇生長良好的海馬神經(jīng)元用2.5%的戊二醛(生理鹽水配制)固定30min,然后用流動(dòng)三蒸水輕輕沖洗樣品,以去除樣品表面的鹽結(jié)晶。剪掉培養(yǎng)皿的皿壁,用502膠水固定于載玻片上,待其在空氣中自然干燥后,置于AFM的掃描器上,進(jìn)行掃描成像,成像模式為大氣環(huán)境下的接觸模式(Contact Mode)。分別取新生48小時(shí)的轉(zhuǎn)基因RFP C57乳鼠、EGFP C57乳鼠(其腦部特異性轉(zhuǎn)入綠色熒光蛋白和紅色熒光蛋白)各一只,在無菌條件下原代混合培養(yǎng)海馬神經(jīng)元細(xì)胞。選擇生長良好的海馬神經(jīng)元在熒光顯微鏡下觀察并拍照。結(jié)果：本實(shí)驗(yàn)中培養(yǎng)的海馬神經(jīng)元生長狀態(tài)良好。(1)利用AFM觀察顯示：正常海馬神經(jīng)元表面光滑,起伏均勻、規(guī)律,神經(jīng)元多為單個(gè)生長,突起之間交織成網(wǎng),相互間有突觸形成。同時(shí),我們還觀察到海馬神經(jīng)元間存在膜性連接以及長程非突觸性突起連接結(jié)構(gòu)；(2)利用熒光顯微鏡觀察顯示：在共培養(yǎng)的細(xì)胞中,發(fā)現(xiàn)兩個(gè)通過膜性連接的海馬神經(jīng)元中的一個(gè)細(xì)胞內(nèi),同時(shí)存在紅色熒光蛋白和綠色熒、光蛋白。結(jié)論：我們在以往的工作中利用AFM觀察到海馬神經(jīng)元的一些利用其它顯微技術(shù)難以發(fā)現(xiàn)的超微結(jié)構(gòu),還發(fā)現(xiàn)了一些用突觸結(jié)構(gòu)難以解釋的連接結(jié)構(gòu),并對其中之一的膜性連接結(jié)構(gòu)進(jìn)行了初步的研究：(1)再次證明原子力顯微技術(shù)是研究細(xì)胞表面超微結(jié)構(gòu)的有用工具；(2)為神經(jīng)元間的膜性和長程非突觸性突起連接提供了更直接的證據(jù)；(3)發(fā)現(xiàn)了神經(jīng)元之間的膜性連接結(jié)構(gòu)可能存在大分子的物質(zhì)的交換或者轉(zhuǎn)運(yùn)。由此推測,神經(jīng)元之間的膜性連接結(jié)構(gòu)可能不僅存在于同一動(dòng)物的生命發(fā)展初期,還有可能在后天一定的刺激條件下形成,以起到信息傳遞甚至物質(zhì)轉(zhuǎn)運(yùn)的作用。
[Abstract]:Objective: to study the surface ultrastructure, connection structure and related functions of hippocampal neurons cultured in primary culture by AFM and transgenic techniques. Materials and methods: C57 pups were collected from newborn mice within 12 hours. Hippocampal neuronal cells were cultured in aseptic condition. Hippocampal neurons with good growth were fixed with 2.5% glutaraldehyde (normal saline) for 30 min. To remove salt crystallization from the surface of the sample, cut off the dish wall of the petri dish, fix it on the slide with 502 glue, and after it is naturally dried in the air, put it on the AFM scanner for scanning imaging. The imaging model was contact mode in the atmospheric environment. One newborn RFP C57 newborn mouse (with brain specific transfer to green fluorescent protein and one red fluorescent protein) was taken from the newborn RFP C57 newborn mouse, which was transformed into green fluorescent protein (GFP) and red fluorescent protein (GFP C57), respectively, in which the brain was transferred specifically to green fluorescent protein (GFP) and red fluorescent protein. Hippocampal neuronal cells were cultured in aseptic condition. Hippocampal neurons with good growth were selected to be observed and photographed under fluorescence microscope. Results: the cultured hippocampal neurons in this experiment were in good growth state. The results showed that the surface of normal hippocampal neurons was smooth. The ups and downs are uniform, regular, neurons are mostly single growth, processes interweave into a network, and synapses form among each other. At the same time, We also observed membranous junctions between hippocampal neurons and long range nonsynaptic neuronal junctions. Using fluorescence microscopy, we observed that in co-cultured cells, Red fluorescent protein and green fluorescence were found in one of two hippocampal neurons via membranous junctions. Conclusion: in our previous work, we have used AFM to observe some ultrastructures of hippocampal neurons that are difficult to find by other microscopic techniques, as well as junctional structures that are difficult to explain by synaptic structures. A preliminary study of the membranous junctional structure of one of them (1) proves that atomic force microscopy (AFM) is a useful tool for studying the ultrastructure of the cell surface, and that atomic force microscopy (AFM) is a useful tool for studying the ultrastructure of the cell surface. This provides more direct evidence of the possible exchange or transport of macromolecular substances in the membranous junction structure between neurons. The membranous junction structure between neurons may exist not only in the early stage of life development of the same animal, but also in the postnatal stimulation, which may play the role of information transmission and even material transport.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R33

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本文編號：1635318